Altered glucose metabolism is a characteristic feature of most cancer cells, yet it is still poorly understood. Because altered metabolism represents a fundamental difference that exists between cancer cells and normal cells, targeting metabolism holds great promise for improved cancer therapy. However, success depends on understanding how metabolic regulation provides an advantage for tumor cells in relevant cancer models. Cancer cells require altered metabolism to efficiently incorporate nutrients such as glucose into biomass to support proliferation. Our understanding of how cancer cells meet these metabolic needs is based primarily on studies of cultured cells, and nutrient levels in vitro are significanty different from those experienced by tumor cells in vivo. Furthermore, cell proliferation rates in tumors can be low, particularly under conditions of metabolic stress, and a separate metabolic program from that used to support proliferation is needed for cells to thrive when nutrients are limited. Our long-term objective is to develop a comprehensive understanding of how cell metabolism is altered to support all stages of cancer progression in vivo. The M2 isoform of pyruvate kinase (PKM2) promotes glucose use for anabolic processes and is the pyruvate kinase isoform found in all human tumors described to date. The PKM1 isoform is found in many differentiated tissues and promotes efficient ATP synthesis from available nutrients. Paradoxically, despite increased glucose metabolism in cancers, PKM2 expression is associated with decreased pyruvate kinase enzyme activity, and complete loss of PKM2 can accelerate tumor growth. We aim to use genetically engineered mouse cancer models and small molecule pyruvate kinase activators in combination with biochemical approaches to understand how pyruvate kinase regulates glucose metabolism in tumors. We also aim to understand how this regulation contributes to cancer initiation and progression and any non-glycolytic PKM2 functions that might be important for tumors. In Specific Aim 1, we will take advantage of mice with conditional pyruvate kinase alleles to define the role of PKM1 and PKM2 in tumor initiation, as well as determine how pyruvate kinase isoform expression influences the growth of established tumors. In Specific Aim 2, we will investigate how the different regulatory properties of PKM2 influence glucose metabolism and how this impacts tumor biology. In Specific Aim 3, we will determine how cells continue to metabolize glucose in the absence of pyruvate kinase. Together, these studies will advance our understanding of glycolytic regulation in cancer cells in vivo. They will also inform how best to target glucose metabolism for improved cancer therapy.